Use of biodegradable microspheres that release an anticancer agent for treating gliobastoma

ABSTRACT

The invention relates to the use of biodegradable microspheres that release a radiosensitizing anticancer agent for producing a medicament to be used simultaneously with, separately from or spread over time with a radiotherapy, for treating glioblastoma. The use of said biodegradable microspheres according to the invention results in a patient survival time of least 90 weeks, a thereapeutically effective concentration being maintained in the parenchymatous area throughout this time. The microspheres used prefereably contain 5-fluorouracile of the tumor, by intratissular injection. The radiothereapy targeting the tumorous mass is dosed at 60 Gy over approximately 6 weeks. The invention also relates to a method for producing the biodegradable microspheres by emulsion-extraction, and to a suspension containing the biodegradable microspheres obtained using this method.

[0001] The present invention relates to the use of biodegradablemicrospheres which release an anticancer agent, for treatingglioblastoma.

[0002] Glioblastoma belongs to the group of rare diseases listed by theNational Organization for Rare Disorders.

[0003] Malignant glial tumors are primary tumors of the central nervoussystem which represent, depending on the series, 13 to 22% ofintracranial tumors. From a histological point of view, two types ofmalignant glial tumor are, in fact, distinguished, anaplasticastrocytomas and glioblastomas, the latter representing the mostundifferentiated form of theses tumors.

[0004] There is currently no effective treatment against malignant glialtumors. The survival time of patients suffering from glioblastoma doesnot exceed one year, even if chemotherapy and radiotherapy are combinedwith surgery.

[0005] The treatment of malignant glial tumors is mainly limited bythree phenomena.

[0006] The first is the existence of a blood-brain barrier (BBB) whichisolates the central nervous system from the rest of the body. This BBBallows only liposoluble molecules which are small in size to pass. Othermolecules must be administered at very high doses in order to reach thecentral nervous system, this being at the cost of considerable systemicside effects.

[0007] The second factor which limits the effectiveness of treatment forglial tumors is the infiltrating nature of these tumors. Since the brainis a highly functional organ, it is impossible to perform on it surgerywhich is exclusive in the carcinological sense of the word. The mostcomplete exeresis possible will only be a macroscopically completeexeresis, leaving a large number of tumor cells infiltrated into thewalls of the exeresis cavity. Many authors have, moreover, shown that90% of malignant glial tumors which are operated on and treated withradiotherapy recur within a distance of two centimeters from the initialtumor site.

[0008] The last factor which limits the effectiveness of treatment forglial tumors is the low therapeutic index. Tumor cells shelter as itwere behind normal tissue which is extremely fragile and sensitive toattacks, caused for example by radiotherapy or by certain anticanceragents. It is thus difficult to destroy the tumor cells withoutdestroying the normal nerve cells.

[0009] The progress achieved in the treatment of glial tumors isinsufficient (Kornblith P L, Walker M, Chemotherapy for malignantgliomas. J. Neurosurg, 68: 1-17, 1988; Shapiro W R, Green S B, Burger PC, Selker R G, VanGilder J C, Robertson J T, Mahaley S M, A randomizedcomparison of intra-arterial versus intravenous BCNU with or withoutintravenous 5-fluorouracil, for newly diagnosed patients with malignantglioma, J. Neurosurg. 76: 772-781, 1992).

[0010] Currently, conventional treatment for glioblastomas, subsequentto surgical resection, is based on external radiotherapy. It does notmake it possible to achieve a survival time of more than one year.Combining radiotherapy with chemotherapy using1-(2-chloroethyl)3-cyclohexyl-1-nitrosourea (BCNU) is effective only onanaplastic astrocytomas. This contributes only modestly since it onlyincreases the percentage of survivors at eighteen months, withoutmodifying the survival time.

[0011] Furthermore, immunotherapy has never established itself in thisarea and gene therapy has yet to prove itself.

[0012] Experiments have been carried out on several techniques aimed atincreasing the local concentration of anticancer agents, such as osmoticrupture of the blood-brain barrier, injection into the cerebrospinalfluid, intracarotid infusion and intratumor administration usingsubcutaneous reservoirs (Tamargo R J and Brem H, Drug delivery to thecentral nervous system, Neurosurgery Quarterly, 2: 259-279, 1992). Noneof these techniques has been able to increase the survival time of thepatients and some have proved to be highly toxic.

[0013] Over the past few years, research in galenic pharmacy has allowedthe development of implantable polymer systems which protect activesubstances against degradation and which allow their controlled localrelease over a given period of time while at the same time decreasingthe systemic side effects. The advantages of these implantable polymersystems have recently prompted several teams to study their use incentral nervous system pathologies (Langer R, Polymer implants for drugdelivery in the brain, J. Controlled Release, 16: 53-60, 1991). Inparticular, such systems implanted into the tumor resection wall ofmalignant gliomas slow down tumor recurrence and prolong patientsurvival. Isolated malignant cells persist around the cavity left afterthe operation, which are responsible for 90% of recurrences, which occurwithin a distance of two centimeters from the operating locus. Withinthis area, the nervous tissue is functional and the blood-brain barrieris still intact, which limits the action of conventional chemotherapyand radiotherapy.

[0014] Diverse implantable polymer systems which release activemolecules have been developed and tested in animals.

[0015] A system of biodegradable wafers which are composed of PCPP-SA(poly[1,3-bis(carboxyphenoxy)propane-co-sebacic acid]) and which releaseBCNU (GLIADEL®) has been developed despite modest results in clinicalstudies (Brem H, Polymers to treat brain tumors, Biomaterials 11:699-701, 1990; Brem H, Mahaley M S, Vick N A, Black K L, Schold S C,Eller T W, Cozzens J W, Kenealy J N, Interstitial chemotherapy with drugpolymer implants for the treatment of recurrent gliomas, J. Neurosurg74: 441-446, 1991; Brem H, Walter K A, Langer R, Polymers as controlleddrug delivery devices for the treatment of malignant brain tumors, Eur JPharm Biopharm, 39 (1): 2-7, 1993; Brem H, Piantadosi S, Burger P C,Walker M, et al., Placebo-controlled trial of safety and efficacy ofintraoperative controlled delivery by biodegradable polymers ofchemotherapy for recurrent glioma, Lancet, 345: 1008-1012, 1995).

[0016] Microspheres which release BCNU have been developed but theresults of studies in animals were relatively unencouraging (Torres A l,Boisdron-Celle M, Benoit J P, Formulation of BCNU-loaded microspheres:influence of drug stability and solubility on the design of themicroencapsulation procedure, J. Microencapsulation, 13: 41-51, 1996;Painbéni T, Venier-Julienne M C, Benoit J P, Internal morphology ofpoly(D,L-lactide-co-glycolide) BNCU-loaded microspheres. Influence ondrug stability, Eur. J. Pharm. Biopharm, 1998, 45, 31-39).

[0017] The subject of the present invention is the use of implantablebiodegradable microspheres which release an anticancer agent, fortreating glioblastoma. The use of these microspheres is combined withradiotherapy and with surgery. After exeresis of the tumor, thebiodegradable microspheres which release an anticancer agent areimplanted into the operating locus by intratissular injection.Radiotherapy is then carried out, within a maximum of seven days afterthe intervention.

[0018] By virtue of using these microspheres, the Applicant hassucceeded, entirely advantageously, in doubling the survival time ofpatients suffering from a glioblastoma. Specifically, the use of themicrospheres according to the invention makes it possible to achieve asurvival time of at least 90 weeks.

[0019] Consequently, the present invention relates to the use ofbiodegradable microspheres which release a radiosensitizing anticanceragent, for manufacturing a medicinal product intended to be usedsimultaneously, separately or spread out over time with radiotherapy,for treating glioblastoma, said microspheres being intended to beimplanted into the operating locus after exeresis of the glial tumor,characterized in that the microspheres containing the anticancer agentare coated with a polymer which delays the release of the anticanceragent and maintains, over time, a therapeutically effectiveconcentration in the parenchymal space with a view to achieving asurvival time for the patient thus treated at least equal toapproximately 90 weeks, preferably approximately 130 weeks, even morepreferably 160 weeks.

[0020] The microspheres used in the context of the invention contain ananticancer agent which is preferably hydrophilic and/or does not crossthe blood-brain barrier. Advantageously, the anticancer agent does notshow central neurotoxicity. This anticancer agent acts preferentially ondividing cells.

[0021] The anticancer agent consists of a radiosensitizing anticancercompound or of a mixture of anticancer compounds containing at least oneradiosensitizing anticancer compound, said anticancer compound(s) being,for example, chosen from 5-fluorouracil (5-FU), platins, such ascarboplatin and cisplatin, taxanes, such as docetaxel and paclitaxel,gemcitabine, VP16, mitomycin, idoxuridine, topoisomerase 1 inhibitors,such as irinotecan, topotecan and camptothecins, nitrosoureas, such asBCNU, ACNU or MCNU, methotrexate, bleomycin, adriamycin, cytoxan andvincristine, immunomodulating cytokines, such as IL2, IL6, IL12 andIL13, and inteferons.

[0022] The anticancer agent is preferably 5-FU.

[0023] 5-FU is a long-standing and well known antimitotic agent. It is ahydrophilic molecule which crosses the blood-brain barrier very poorlyand its activity is therefore increased by local administration (BourkeR S, West C R, Chheda G et al., Kinetics of entry and distribution of5-fluorouracil in CSF and brain following intravenous injection inprimate, Cancer Res, 33: 1735-1746, 1973; Gerosa M A, Dougherty D V,Wilson C B, Rosenblum M L, Improved treatment of a brain tumor model,Part 2: Sequential therapy with BCNU and 5-fluorouracil, J. Neurosurg.58: 368, 1983; Kotsilimbas D G, Karpf R, Meredith S, Scheinberg L C,Evaluation of parenteral 5-FU on experimental brain tumors, Neurology,16: 916-918, 1966; Levin V A, Edwards M S, Wara W M, Allen J, Ortega J,Vestnys P, 5-fluorouracil and1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) followed byhydroxyurea, misonidazole and irradiation for brain stem gliomas: apilot study of the brain tumor research center and the children cancergroup, Neurosurgery, 14: 679-681, 1984; Oda Y, Tokuriki Y, Tsuda E,Handa H, Kieler J, Trial of anticancer pellet in malignant braintumours, 5-FU and urokinase embedded in silastic. Proceeding of the 6thEuropean Congress of Neurosurgery, Acta neurochirurgica, Suppl. 28:489-490, 1979; Penn R D, Kroin J S, Harris J E, Chiu K M, Braun D P,Chronic intratumoral chemotherapy of a rat tumor with cisplatin andfluorouracil, Appl. Neurophysio, 46: 240-244, 1983; Shapiro W R, Studieson the chemotherapy of experimental brain tumors: Evaluation of1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, vincristine and5-fluorouracil, J. Nat. Cancer Institute, 46(2), 359-368, 1971; ShapiroW R, Green S B, Burger P C, Selker R G, VanGilder J C, Robertson J T,Mahaley S M, A randomized comparison of intra-arterial versusintravenous BCNU with or without intravenous 5-fluorouracil, for newlydiagnosed patients with malignant glioma, J. Neurosurg, 76: 772-781,1992; Soloway A H, Mark V H, Dukat E G et al., Chemotherapy of braintumors. I-Transplanted murine ependymoblastomas, Cancer Chemother Rep.,36: 1-4, 1964).

[0024] The activity of 5-FU is thus increased by sustainedadministration. 5-FU is an agent which acts by interfering with nucleicacid synthesis. Studies have shown that only 30 to 50% of the cells of amurine malignant glioma (L9), and 14 to 44% of the cells of a humanmalignant glioma, are dividing at any given moment. In addition, thedurations of the glioblastoma cell cycle are long (20 hours for the L9glioma, from 3 to 7 days for human glioblastoma) . Now, 5-FU clearancein the plasma is rapid (30 min half-life) (Neuwelt E A, Barnett P A,Frenkel E P, Chemotherapeutic agent permeability to normal brain anddelivery to avian sarcoma virus-induced brain tumors in the rodent:observation on problems of drug delivery, Neurosurgery, 14: 154-160,1984). 5-FU cannot therefore destroy, via systemic administration orlocal injection, a large number of malignant cells.

[0025] 5-FU is essentially active on tissues which undergo rapid renewaland is exceptionally neurotoxic. 5-FU intervenes in the synthesis ofnucleic acids, which tissues with rapid growth particularly need inorder to ensure their proliferation and regeneration. This is, ofcourse, not the case of cerebral tissue, where mitoses are rare in thenormal state and occur only among the glial population. The toxiceffects of 5-FU which limit its administration via the general route areessentially hematological and gastrointestinal. While rare neurologicalside effects of 5-FU have been published, their etiopathogeny, which isrelatively unknown, is probably multifactorial (blockage of the Krebscycle by a 5-FU catabolite or exacerbation of a preexisting thiaminedeficiency) (Aoki N, Reversible leukoencephalopathy caused by5-fluorouracil derivatives, presenting as akinetic mutism, Surg Neurol,25: 279-282, 1986; Moore D H, Fowler W C, Crumpler L S, 5-fluorouracilneurotoxicity, case report, Gynecol Oncology, 36: 152-154, 1990).

[0026] Finally, 5-FU is radiosensitizing (Koutcher J A, Alfieri A A,Thaler H et al., Radiation enhancement by biochemical modulation and5-FU, Int. J. Radit, Biol. Phys., 39: 1145-1152, 1997). The superiorityof the combination 5-FU/radiotherapy in each of these isolatedtreatments was demonstrated as early as the 1960s on animal models andon tumor cells in vitro (Bagshaw M, A possible role of potentiation inradiation therapy, Amer J. Roentgenol, 85: 822-833, 1961; Vietti T,Eggerding F, Valeriote F, Combined effect of X-radiation and5-fluorouracil on survival of transplanted leukemic cells, J. Natl.Inst., 47: 865-870, 1971). This synergy is thought to be due tosynchronization of the tumor cell population and to a decrease inmechanisms of cellular repair through the 5-FU. The combination ofradiotherapy and antipyrimidine (5-FU or BrudR) has already beenattempted in humans (Goffman T E, Dachowski L J, Bobo H et al., Longterm follow-up on national cancer institute phase I/II study ofglioblastoma multiforme treated with iododeoxyuridine andhyperfractionated irradiation, J. Clinical Oncology, 10: 264-268, 1992).

[0027] The absence of clear-cut effectiveness may once again beexplained by the systemic route of administration of the drugs.

[0028] When the anticancer agent is 5-FU, the concentration ofanticancer agent in the cerebrospinal fluid, which mirrors theconcentration in the parenchymal space, is between 3 and 20 ng/ml.

[0029] In order to limit the neurotoxicity of the anticancer agentcontained in the microspheres used in the context of the invention, aneuroprotective compound may advantageously be added to said anticanceragent. This neuroprotective compound is, for example, chosen frompeptide growth factors, such as NGF or BDNF.

[0030] The biodegradable microspheres used in the context of theinvention are coated with a polymer which delays the release of theanticancer agent and maintains, in the parenchymal space, atherapeutically effective concentration for a period of time of at leastthree weeks, preferably of at least four weeks.

[0031] The polymer is chosen from ethylcellulose, polystyrene,poly(ε-caprolactone), poly(d,l-lactic acid) and poly(d,l-lacticacid-co-glycolic acid).

[0032] The polymer is preferably poly(d,l-lactic acid-coglycolic acid),or PLAGA, which is a biodegradable polymer permitted in the formulationof sustained-release galenic preparations (unlike PCPP-SA, which is notapproved for large-scale clinical use).

[0033] The poly(d,l-lactic acid-co-glycolic acid) is preferably 50:50PLAGA (i.e. containing an equal amount of lactic acid and of glycolicacid), for example Resomer® RG 506 supplied by BI Chimie, France, whichhas a weight-average molecular mass equal to 72 000, a polydispersityindex equal to 1.8 and an inherent viscosity of 0.80 dl/g (0.1% solutionof polymer in chloroform at 25° C.).

[0034] PLAGA is a hydrophobic copolymer, the degradation of which,caused by a hydrolysis reaction, gives rise to two normal biologicalsubstrates, lactic acid and glycolic acid, which are metabolized at theend of aerobic glycolysis to CO₂ and H₂O. Studies, which are alreadylong-established, have shown that the respiratory pathway is the mainpathway of elimination of these two substrates. The rate ofbiodegradation of PLAGA depends on the respective proportions of lacticacid and glycolic acid. PLAGA is completely biocompatible and causes amoderate foreign body reaction (Visscher G E, R L Robinson, H V Mauding,Fong J W, Pearson J E, Argentieri G J, Biodegradation of and tissuereaction to 50:50 poly(DL-lactide-co-glycolide) microcapsules, J.Biomed. Mat. Res. 19: 345-365, 1985). PLAGA is a constituent element ofsurgical sutures (Frazza E J, Schmidt E E, A new absorbable suture, J.Biomed. Mater. Res., 5:43-58, 1971) and of subcutaneously implantablegalenic forms (Jalil R, Nixon J R, Biodegradable poly(lactic acid) andpoly(lactide-co-glycolide) microcapsules: problems associated withpreparative techniques and release properties (Review), J.Microencapsulation, 7: 297-325, 1990). It has been demonstrated that50:50 PLAGA microspheres may be sterilized by γ-irradiation, and that,once implanted by stereotaxy into the brain of a rodent, they arecompletely biodegraded within two months, causing only moderate reactionof the nonspecific astrocyte and histiocyte type (Menei P, Daniel V,Montero-Menei C, Brouillard M, Pouplard-Barthelaix A, Benoit J P:Biodegradation and brain tissue reaction topoly(DL-lactide-co-glycolide) microspheres, Biomaterials 14: 470-478,1993; Menei P, Croue A, Daniel V, Pouplard-Barthelaix A, Benoit J P:Fate and biocompatibility of three types of microspheres implanted intothe brain, J. Biomed Mat Res, 28, 1079-1085, 1994). The latter resulthas since been confirmed by Kou J H, Emmett C, Shen P et al., Bioerosionand biocompatibility of poly(d,l-lactic-co-glycolic acid) implants inbrain, J Control Release, 43, 123-130, 1997.

[0035] The biodegradable microspheres of the invention preferably have amean diameter of 48±20 μm, preferably 46±7 μm. They contain 15 to 35% byweight of anticancer agent, preferably from 19 to 27% of 5-FU, even morepreferably 20% of 5-FU, and 65 to 85% by weight of polymer.

[0036] In the context of the present invention, the 50:50 PLAGAmicrospheres vehiculing 5-FU are particularly preferred.

[0037] In vitro, the 50:50 PLAGA microspheres vehiculing 5-FU canrelease 5-FU for 21 days. In vivo, implanted subcutaneously in rabbits,these microspheres make it possible to obtain a plateau concentration of5-FU in the plasma for 23 days. Still in vivo and in the rodent brain,the crystals of 5-FU are visible in the microspheres at least up to the19th day. In rabbits, after intracerebral implantation of PLAGA-5-FUmicrospheres, (7 mg/kg of 5-FU), no trace of 5-FU is detected in theserum, which leads to the suggestion that there has been virtually nopassing of the drug into the systemic circulation.

[0038] After intracerebral implantation of PLAGA-5-FU microspheres inrodents, at a total dose of 17 mg/kg of 5-FU, no sign of systemictoxicity, nor any sign of clinical or histological neurotoxicity, wasobserved. At the fractionated dose of 24 Gy, the combination of 5-FUmicrospheres/cerebral radiotherapy is completely tolerated (Menei P,Vectorisation dans le SNC par implantation stéréotaxique de microspheres[Vectorization in the CNS by stereotactic implantation of microspheres],These d'Université en Sciences Pharmaceutiques [University doctorate inpharmaceutical sciences], Université d'Angers [University of Angers],1995). Finally, these microspheres implanted by stereotaxy into amalignant glioma developed in rats (C6 glioma) significantly decreasemortality (Menei P, Boisdron-Celle M, Croue A, Guy G, Benoit J P, Effectof stereotactic implantation of biodegradable 5-fluorouracil-loadedmicrospheres in normal and C6-glioma bearing rats, Neurosurgery, 39:117-124, 1996).

[0039] Advantageously, the microspheres are suspended in a sterilesolution, the suspension being injected into the walls of the operatinglocus, after exeresis of the tumor.

[0040] The sterile solution preferably contains

[0041] between 1 and 1.5%, preferably 1.25% weight/volume, of aviscosity modifier, for example sodium carboxymethylcellulose,

[0042] between 0.5 and 1.5%, preferably 1%, of a surfactant, for examplepolysorbate 80®, and

[0043] between 3.5 and 4.5%, preferably 4%, of an isotonicity agent, forexample mannitol.

[0044] The microspheres are preferably suspended extemporaneously justbefore injection. The suspension preferably contains 3 ml of the sterilesolution described above and 700 to 800 mg of biodegradablemicrospheres.

[0045] After confirmation of the diagnosis of glioblastoma andmacroscopic exeresis of the glial tumor, the suspension of microspheresis implanted into the walls of the operating locus, at a depth of atleast two centimeters, preferably between 2 and 3 centimeters, at leastevery cm².

[0046] When the anticancer agent is 5-FU, the total dose of suspensioninjected corresponds to an amount of 5-FU of between 50 and 200 mg.

[0047] The radiotherapy is focussed on the tumor volume, the volumeirradiated encompasses the preoperative tumor with a margin of at leasttwo centimeters in all directions, and a total dose of between 50 Gy and60 Gy is applied.

[0048] The radiotherapy is preferably initiated between the second andseventh days after the operation. A total dose of between 50 Gy and 60Gy is spread out over a period of time of between 4 and 8 weeks, forexample at a rate of 5 fractions per week.

[0049] The radiotherapy is preferably carried out with a total dose of60 Gy for approximately six weeks, preferably again at a rate of fivefractions per week for 6.5 weeks.

[0050] After having injected the microspheres just after exeresis of thetumor, one or more new injections of microspheres may be performed bystereotaxy in the event of recurrence of the tumor.

[0051] The microspheres used in the context of the invention may beprepared using an emulsification-extraction technique according to avariation of the method described by Boisdron-Celle M, Menei P, Benoit JP: Preparation of biodegradable 5-fluorouracil-loaded microspheres, JPharm Pharmacol, 47, 108-114, 1995.

[0052] The present invention also relates to a method for preparing themicrospheres containing an anticancer agent, coated with a polymer usedin the context of the invention. The major steps of this method consistin preparing an organic phase in which the anticancer agent and thepolymer are dispersed in an organic solvent. The organic phase and anaqueous phase are emulsified, and then the organic solvent is extractedby adding water. Finally, the suspension of microspheres obtained isfiltered.

[0053] The method of the invention is first of all characterized in thatthe anticancer agent is dispersed in the organic solvent, with vigorousstirring, before the polymer is added.

[0054] Depending on the adaptations made to the method of the prior art,the active principle is ground in a planetary ball mill. The size of thecrystals obtained is between 15 and 50 μm. The size of the crystals tobe encapsulated and their dispersion are, in fact, essential criteriafor controlling the degree of encapsulation and the in vitro releasekinetics.

[0055] The active principle is then dispersed in an organic solvent,preferably dichloromethane, in a round-bottomed tube, with stirringusing a homogenization rod, before the polymer is added.

[0056] The homogenization gives a homogeneous suspension, attenuates thedifferences from one grinding batch to another and reduces the size ofthe crystals of the active principle.

[0057] The organic phase is prepared in a solvent without cosolvent. Theabsence of cosolvent slows down the precipitation of the polymer duringthe emulsification phase, such that the particles obtained are lessporous.

[0058] The active principle dispersion is transferred into a firstreactor.

[0059] The polymer is added in a proportion by mass of between 8 and13%, preferably equal to 11%. The organic phase obtained is maintainedat room temperature with constant stirring for 2 to 4 hours and then forapproximately 15 minutes at a temperature of between 1 and 5° C.,preferably equal to 2° C. A longer period of stirring of the organicphase at room temperature ensures total solubilization of the polymer inthe solvent.

[0060] The aqueous phase is prepared in a second reactor, preferablymaintaining it at the same temperature as the organic phase, preferablyat 2° C. Reduction of the temperature of the aqueous phase and of theorganic phase causes an increase in their viscosity and an increase inthe degree of encapsulation. The aqueous phase is, for example, anaqueous 10% PVA solution.

[0061] Two jacketed reactors are used and a coolant liquid circulates inseries in the two reactors. The temperature of the organic and aqueousphases is advantageously identical, preferably equal to 2° C., when thetwo phases are mixed together. Good control of the temperatureeffectively conditions the particle size, the rate of dissolution of theactive principle and the extraction speed of the solvent all at once.

[0062] The organic phase is transferred from the first reactor into thesecond. The aqueous phase/organic phase proportion by volume is between80/3 and 120/3, preferably equal to 100/3.

[0063] The emulsion obtained is stirred for at least 3 minutes,preferably for 3 to 6 minutes, even more preferably for 5 minutes. Thechoice of this period of time is directly correlated with the releasekinetics and in particular the “burst” effect over 24-48 hours.

[0064] The absence of cosolvent coupled with a sufficient emulsificationtime allows dissolution of the active principle which is at the surfaceor poorly coated, such that the release kinetics in the initial phaseare better controlled.

[0065] Water is added to the emulsion, in an emulsion/water ratio byvolume of between 1/4 and 1/2, preferably equal to 1/3, in order toextract the organic solvent. The temperature of the extraction water isbetween 1 and 5° C., preferably equal to 4° C.

[0066] The emulsification and extraction steps are carried out in thesame reactor so as to limit the variability from one batch to anotherand to save time. The temperature of the extraction water is low, so asto limit an excessive dissolution of the active principle.

[0067] The suspension of microspheres obtained is mixed for a fewminutes and then filtered under an inert atmosphere. Working under aninert atmosphere makes it possible to limit the risks of contaminationof the product.

[0068] The microspheres, which may be obtained according to the methoddescribed above, are advantageously lyophilized.

[0069] 10 ml of sterile water are added to 2 to 5 g of microspherepowder (filtercake). This mixture is frozen at −40° C. and thenintroduced into the freeze-dryer. The lyophilization lasts 18 hours. Atthe end of the operation, the secondary drying temperature should bemaintained below 10° C.

[0070] The microspheres should be stored at +40° C., even when dry.

[0071] The present invention also relates to a suspension consisting ofa sterile solution containing 1 to 1.5% mass/volume of a viscositymodifier, 0.5 to 1.5% of a surfactant and 3.5 to 4.5% of an isotonicityagent, and biodegradable microspheres which release an anticancer agent,which are coated with a polymer and which are described above,optionally obtained according to the method described above, theproportion of the microspheres representing 200 to 300 mg/ml of sterilesolution, preferably 230 to 270 mg/ml.

[0072] These microspheres preferably consist of 15 to 35% by weight ofanticancer agent and 65 to 85% by weight of polymer.

[0073] The polymer is advantageously poly(d,l-lactic acid-co-glycolicacid) preferably containing an equal amount of lactic acid and glycolicacid.

[0074] The sterile solution preferably contains 1.25% weight/volume ofsodium carboxymethylcellulose, 1% of polysorbate 80 and 4% of mannitol.

[0075] The present invention is illustrated in a nonlimiting manner bythe following examples.

EXAMPLE 1

[0076] Microspheres prepared using the emulsification-extraction ofsolvent technique, according to a variation of the method described byBoisdron-Celle M, Menei P and Benoit J P (Preparation of biodegradable5-fluorouracil-loaded microspheres, J Pharm Pharmacol, 47, 108-114,1995).

Grinding of 5-FU

[0077] 5-FU is ground in a planetary ball mill such as the Pulverisette7 (Fritsch). 8.5 g of 5-FU are introduced into a beaker containing 7beads. The grinding lasts 10 minutes at speed 7. The powder is recoveredunder a laminar flow hood. The crystals obtained are between 15 and 50μm in size and are divided into two fractions: a fine fraction (with aparticle size of less than 1 μm) and a coarse fraction (greater than 30μm).

Dispersion of 5-FU in the organic solvent

[0078] The ground 5-FU is dispersed in 45 ml of dichloromethane withstirring using a homogenizer such as an Ultra-Turrax machine for 3minutes at 13 500 rpm, in a round-bottomed tube.

Preparation of the organic phase

[0079] The 5-FU dispersion is transferred into a 150 ml jacketed cooledreactor. The PLAGA is added thereto such that the PLAGA/dichloromethaneproportion is equal to 11%. The organic phase is stirred with a paddle,at 450 rpm, for 4 hours at 20° C., and then for 15 minutes at 2° C. Thetemperature in the reactor is kept constant to within 0.1° C. with theaid of a cryostat.

Preparation of the emulsion

[0080] 1 500 ml of an autoclaved aqueous 10% PVA solution are preparedand maintained at 2° C. in a 6 liter jacketed cooled reactor. Theorganic phase is then transferred into this reactor by opening a basevalve on the first reactor. The organic phase is poured over 5 to 10 sonto the aqueous phase, which is stirred using a paddle rotating at 375rpm. The aqueous phase/organic phase proportion by volume is equal to100/3.

[0081] The emulsion is stirred for 4 minutes and 45 s.

Extraction

[0082] When the emulsion is ready, 4.5 1 of extraction water at 4° C.are poured onto the emulsion in an emulsion/water ratio by volume equalto 1/3. The extraction lasts 2 minutes.

Filtration

[0083] The entire contents of the second reactor are transferred, viathe base, into a stainless steel tank and then placed under a pressureof nitrogen. The suspension is filtered through a filter with a porediameter equal to 3 μm.

[0084] After passing the entire suspension through the filter, thefiltercake is washed twice with 3 liters of sterile water.

[0085] The degree of encapsulation of the anticancer agent in themicrospheres obtained is 20%. After sieving, desorption of thedichloromethane is performed in an oven for 48 hours. The microspheresare then packaged and sterilized by γ-irradiation at 19 kGy. Furthermonitoring of the degree of encapsulation is carried out aftersterilization. An assay of the residual traces of solvent is thencarried out. A residual level of dichloromethane of 0.5% isadvantageously detected. The sterility and in vitro release kinetics ofthe microspheres obtained are monitored.

[0086] The microspheres obtained have an active principle content of23±3.5%.

[0087] Several batches are produced according to the protocol describedabove and a mean particle size of 48±20 μm is calculated over thepopulation of all of the batches, equivalent to 46±7μm (mean of themeans of the batches prepared).

[0088] The microspheres obtained have an active principle content of23±3.5% and a mean size of 48±20μm.

EXAMPLE 2

[0089] Microspheres are prepared using the emulsification-extraction ofsolvent technique of example 1.

Grinding of 5-FU

[0090] The procedure as in example 1 is carried out and 4 g of 5-FU areground.

[0091] The crystals obtained are between 15 and 50 μm in size and aredivided into two fractions: a fine fraction (with a particle size ofless than 1 μm) and a coarse fraction (greater than 30 μm).

Dispersion of the 5-FU in the organic solvent

[0092] The ground 5-FU is dispersed in 40 ml of dichloromethane withstirring using a homogenizer such as an Ultra-Turrax machine for 3.5minutes at 13 500 rpm, in a round-bottomed tube.

[0093] The organic phase and the emulsion are prepared as in example 1.

[0094] The extraction and filtration are carried out as in example 1.

[0095] Characteristics of the microspheres obtained. 5-FU content: 22%

[0096] Size: 46±7 μm

[0097] Burst effect at 24 h after radiosterilization at

[0098] 19 kGy: 40±4%.

EXAMPLE 3

[0099] A phase I/II open pilot clinical study is carried out with the50:50 PLAGA/5-FU microspheres of example 1.

[0100] The microspheres obtained are suspended extemporaneously in asolution containing

[0101] 1.25% weight/volume of sodium carboxymethyl-cellulose (Cooper),

[0102] 1% of polysorbate 80,

[0103] 4% of mannitol, and

[0104] a sufficient amount of water for injectable preparation, toobtain a total volume of 3 ml.

[0105] The solution is presterilized by autoclaving at 121° C. for 20minutes, and then by radiosterilization by gamma-irradiation at a doseof between 5 kGy and 25 kGy, preferably 19 kGy.

[0106] The preparation of this suspension is delicate since theformation of bubbles has to be avoided. The suspension is injectedimmediately after it has been prepared, since the microspheres have atendency to sediment in the syringe and block it.

[0107] The microsphere suspension is implanted into the walls of theoperating locus, after macroscopic exeresis of the glial tumor, to adepth of between two and three centimeters, every cm , at a rate of 100μl per injection.

[0108] The injection is performed with a 1 ml syringe and a catheter(Insyte® Vialon™) of 18 ga (1.3×45 mm), the metal mandrel of which isremoved so as to retain only the unbeveled foam-tipped plastic catheterto inject the suspension into the cerebral tissue. As many 1 ml syringesas necessary are used. Injection of the suspension with the beveledneedle introduces the risk of hematoma and backflow of the microspheres.The catheter should be small enough in diameter so as not to traumatizethe cerebral tissue and large enough so as not to be blocked by themicrosphere suspension.

[0109] The injection should be performed very gently and the cathetershould remain in place for a few minutes before being removed, so as toavoid backflow of the microspheres. A 1 cm² fragment of resorbablehemostatic compress (Surgical® or Spongel®) is applied to the injectionsite.

[0110] The patients included in the study are between 18 and 68 yearsold, have no neoplastic history, have a Karnofsky index of greater than60, have a clinical history and imaging evoking a sustentacularglioblastoma, have undergone a macroscopically complete exeresis, andtheir intraoperative histological examination (performed according toWHO criteria: necrosis, vascular proliferation, nuclear pleomorphism andmitotic activity) confirms the diagnosis of glioblastoma.

[0111] The criteria for excluding patients are as follows: metabolicdeficiency, pregnancy, other prior cancer pathology.

[0112] Three groups were initially envisioned to study the effects ofincreasing doses of 5-fluorouracil: in chronological order, 70, 132 and264 mg, the treatment of the following group being started aftertolerance of the treatment by the group undergoing testing has beenobserved.

[0113] Due to the occurrence of Grade II neurological toxicity in apatient who received 132 mg of 5-FU, and according to the rules forstopping the protocol, the therapeutic escalation was stopped and thesubsequent patients received the same dose of 132 mg.

[0114] Conventional external radiotherapy (focused on the tumor volumeassessed on preoperative MRI with an energy of 10 MV) is initiatedbetween the second and seventh days after the operation. A total dose of60 Gy in 33 fractions of 1.8 Gy, at a rate of 5 fractions over 6.5weeks, is applied. The volume irradiated encompasses the preoperativetumor with a margin of at least two centimeters in all directions.

[0115] The patients are monitored clinically and radiologically: a scanat 72 hours to confirm the macroscopically complete exeresis and aclinical assessment are carried out on D10, D20 and D30. MRI is carriedout on D10 and D30. Finally, an assay of the 5-FU in the blood and theCSF is performed at 72 hours and on D10, D20 and D30. The toxicity(neurological, hematological, mucous membrane and cardiological) isassessed in grades on criteria derived from those of the WHO. After onemonth has passed, the patients are monitored clinically every two monthsand undergo MRI every three months.

[0116] Mild postoperative anemia, hyperleukocytosis and mild lymphopeniawere observed in all the patients.

[0117] The pharmacological study confirmed the sustained release of 5-FUin the cerebrospinal fluid (CSF) for more than 30 days, and a transientpassage, at a lower level, of the molecule into the systemiccirculation. Significant concentrations of 5-FU are still present in theCSF one month after implantation.

[0118] The profiles of release of 5-FU in the CSF show a peak on thetenth and on the twentieth day, respectively, for doses of 70 and 132mg. The level of 5-FU in the plasma was not detectable from the tenthday in half the patients.

[0119] The systemic tolerance is excellent in all the patients treated.No change in the chemistry or cellularity of the CSF was observed. Theappearance of a cerebral edema during radiotherapy in a patient treatedwith 132 mg did not permit escalation of the dose to be continued.

[0120] Eight patients, including four males and four females, with anaverage age of 48.5 and a Karnofsky index of greater than 90, were thusincluded in the study. The first group of three received a dose of 70mg, and the second group of five received 132 mg.

[0121] The preliminary results regarding survival could not beinterpreted statistically due to the small number of patients. Theywere, however, very encouraging. At the final assessment, in the firstgroup treated (70 mg), the three patients died at 61, 114 and 125 weeks.It should be noted that the patient who died at 114 weeks died ofpulmonary metastases of the glioblastoma. In the second group treated(132 mg), three patients died at 31, 59 and 82 weeks and two were stillin remission at 159 and 172 weeks, at the date of drafting of thesepreliminary results.

[0122] The survival median for the patients is 98 weeks (it is 50.6weeks in the literature for patients satisfying the same criteria(Devaux B C, O'Fallon J R, Kelly P J, Resection, biopsy and survival inmalignant glial neoplasms, J Neurosurg, 78: 767-775, 1993). Five out ofeight patients, i.e. 62%, were alive at 18 months, whereas, in theliterature, for patients satisfying the inclusion criteria of thisstudy, the survival at 18 months is 20% (Devaux B C, O'Fallon J R, KellyP J, Resection, biopsy and survival in malignant glial neoplasms, JNeurosurg, 78: 767-775, 1993).

1. The use of biodegradable microspheres which release aradiosensitizing anticancer agent, for manufacturing a medicinal productintended to be used simultaneously, separately or spread out over timewith radiotherapy, for treating glioblastoma, said microspheres beingintended to be implanted into the operating locus after exeresis of theglial tumor, characterized in that the microspheres containing theanticancer agent are coated with a polymer which delays the release ofthe anticancer agent and maintains, over time, a therapeuticallyeffective concentration in the parenchymal space with a view toachieving a survival time for the patient thus treated at least equal toapproximately 90 weeks, preferably approximately 130 weeks, even morepreferably 160 weeks.
 2. The use as claimed in claim 1, characterized inthat the anticancer agent is hydrophilic and/or does not cross theblood-brain barrier.
 3. The use as claimed in claim 1 or 2,characterized in that the anticancer agent does not show centralneurotoxicity.
 4. The use as claimed in one of the preceding claims,characterized in that the anticancer agent consists of aradiosensitizing anticancer compound or of a mixture of anticancercompounds containing at least one radiosensitizing anticancer compound,said anticancer compound(s) being, for example, chosen from5-fluorouracil (5-FU), platins, such as carboplatin and cisplatin,taxanes, such as docetaxel and paclitaxel, gemcitabine, VP16, mitomycin,idoxuridine, topoisomerase 1 inhibitors, such as irinotecan, topotecanand camptothecins, nitrosoureas, such as BCNU, ACNU or MCNU,methotrexate, bleomycin, adriamycin, cytoxan and vincristine,immunomodulating cytokines, such as IL2, IL6, IL12 and IL13, andinteferons.
 5. The use as claimed in claim 4, characterized in that theanticancer agent is 5-fluorouracil.
 6. The use as claimed in claim 5,characterized in that the concentration of 5-FU in the cerebrospinalfluid, which mirrors the concentration in the parenchymal space, isbetween 3 and 20 ng/ml.
 7. The use as claimed in one of the precedingclaims, characterized in that a neuroprotective compound chosen frompeptide growth factors, such as NGF or BDNF, is added to the anticanceragent.
 8. The use as claimed in one of the preceding claims,characterized in that the polymer coating the microspheres which slowsdown the release of the anticancer agent maintains, in the parenchymalspace, said therapeutically effective concentration for at least threeweeks, preferably at least four weeks.
 9. The use as claimed in one ofthe preceding claims, characterized in that the polymer is chosen fromethylcellulose, polystyrene, poly(ε-caprolactone), poly(d,l-lactic acid)and poly(d,l-lactic acid-coglycolic acid).
 10. The use as claimed in oneof the preceding claims, characterized in that the polymer which coatsthe microspheres is a poly(d,l-lactic acid-co-glycolic acid).
 11. Theuse as claimed in claim 10, characterized in that the polymer whichcoats the microspheres is a poly(d,l-lactic acid-co-glycolic acid)containing an equal amount of lactic acid and of glycolic acid.
 12. Theuse as claimed in one of the preceding claims, characterized in that themicrospheres have a mean diameter of 48±20 μm, preferably 46±7 μm. 13.The use as claimed in one of the preceding claims, characterized in thatthe microspheres contain 15 to 35% by weight of anticancer agent and 65to 85% by weight of polymer.
 14. The use as claimed in claims 5 and 13,characterized in that the microspheres contain 19 to 27% of 5-FU,preferably 20% of 5-FU.
 15. The use as claimed in one of the precedingclaims, characterized in that the microspheres are suspended in asterile solution, the suspension obtained being intended to be injectedinto the walls of the operating locus after exeresis of the glial tumor,and in that said sterile solution contains from 1 to 1.5% mass/volume ofa viscosity modifier, 0.5 to 1.5% of a surfactant and 3.5 to 4.5% of anisotonicity agent.
 16. The use as claimed in claim 15, characterized inthat the solution contains 1.25% weight/volume of sodiumcarboxymethylcellulose, 1% of polysorbate 80 and 4% of mannitol.
 17. Theuse as claimed in claim 15 or 16, characterized in that the suspensionof microspheres is injected into the walls of the operating locus, to adepth of at least two centimeters, preferably between 2 and 3centimeters, at least every cm².
 18. The use as claimed in claim 5,characterized in that the total dose of 5-FU injected is between 50 and200 mg, preferably 130 mg.
 19. The use as claimed in one of thepreceding claims, characterized in that the radiotherapy is focused onthe tumor volume, in that the volume irradiated encompasses thepreoperative tumor with a margin of at least two centimeters in alldirections and in that a total dose of between 50 Gy and 60 Gy isapplied.
 20. The use as claimed in one of the preceding claims,characterized in that the radiotherapy is preferably initiated betweenthe second and seventh day after the operation.
 21. The use as claimedin one of the preceding claims, characterized in that the radiotherapyis carried out with a total dose of 60 Gy for approximately six weeks.22. The use as claimed in one of claims 15 to 17 or 19 to 21,characterized in that the suspension of microspheres is injected bystereotaxy on one or more occasions in the event of recurrence of thetumor.
 23. A method for preparing biodegradable microspheres containingan anticancer agent coated with a polymer, by emulsification-extraction,which consists in dispersing the polymer and the anticancer agent in anorganic solvent, in mixing the organic phase obtained with an aqueousphase so as to obtain an emulsion, in extracting the organic solvent byadding water, and then in filtering the suspension of microspheresobtained, characterized in that the anticancer agent is dispersed in theorganic solvent, with vigorous stirring, before the polymer is added.24. The method as claimed in claim 23, characterized in that the organicsolvent is dichloromethane.
 25. The method as claimed in claim 23 or 24,characterized in that the aqueous phase and the organic phase have anidentical temperature, preferably equal to approximately 2° C., whenthey are mixed.
 26. The method as claimed in one of claims 23 to 25,characterized in that the organic phase contains 11% of polymer.
 27. Themethod as claimed in one of claims 23 to 26, characterized in that theaqueous phase/organic phase proportion is equal to 100/3.
 28. The methodas claimed in one of claims 23 to 27, characterized in that the emulsionconsisting of the aqueous phase and of the organic phase is mixed for atleast 3 minutes.
 29. The method as claimed in one of claims 23 to 28,characterized in that the water required to extract the organic solventis added in a proportion such that the emulsion/water ratio by volume isequal to 1/3.
 30. The method as claimed in one of claims 23 to 29,characterized in that the temperature of the water required to extractthe organic solvent is 4° C.
 31. The method as claimed in one of claims23 to 30, characterized in that the anticancer agent is ground beforebeing dispersed in the organic solvent, such that the size of thecrystals of the anticancer agent is between 15 and 50 μm.
 32. The methodas claimed in one of claims 23 to 31, characterized in that after theextraction water has been added, the suspension obtained is filteredunder an inert atmosphere.
 33. The method as claimed in one of claims 23to 32, characterized in that the microspheres are lyophilized.
 34. Asuspension consisting of a sterile solution containing 1 to 1.5%mass/volume of a viscosity modifier, 0.5 to 1.5% of a surfactant and 3.5to 4.5% of an isotonicity agent, and of biodegradable microspheres whichrelease an anticancer agent, which are coated with a polymer, theproportion of the microspheres representing 200 to 300 mg/ml of sterilesolution.
 35. The suspension as claimed in claim 34, characterized inthat the microspheres consist of 15 to 35% by weight of anticancer agentand of 65% to 85% by weight of polymer.
 36. The suspension as claimed inclaim 34 or 35, characterized in that the polymer is poly(d,l-lacticacid-co-glycolic acid) preferably containing an equal amount of lacticacid and of glycolic acid.
 37. The suspension as claimed in one ofclaims 34 to 36, characterized in that the sterile solution contains1.25% weight/volume of sodium carboxymethyl-cellulose, 1% of polysorbate80 and 4% of mannitol.